Macrophages Alter Metabolism of Engineered Cardiac Tissue in Infarct Conditions

Introduction: : Myocardial infarction (MI) is the most prevalent cause of morbidity and mortality among cardiovascular diseases. MI occurs when coronary blood supply to a region of the myocardium is obstructed, causing ischemic injury and cardiomyocyte death. When blood supply is reestablished, the reintroduction of oxygen causes a burst of respiration and, paradoxically, further damage. While the mechanisms underlying this phenomenon are complex, it is increasingly evident that immune cells – and macrophages in particular – are critical modulators of post-MI outcomes.
Macrophages (Mφ) drive outcomes in myocardial infarction models in rodents depending largely on their developmental origin. Mφ respond in stress conditions such as MI by clearing debris and secreting cytokines to drive inflammation and subsequent tissue remodeling. Mφ also regulate the metabolism of cardiomyocytes. Despite this seeming centrality, Mφ have historically been omitted from engineered models of the human myocardium, limiting the translatability of these models.
Macrophages have recently been incorporated into engineered models of the human myocardium to improve maturity in these tissues, but their role in disease states – particularly MI – has not been assessed. We propose that because of their importance in not only MI outcomes, but in metabolic regulation of the myocardium, that Mφ exert critical control over cardiomyocyte metabolic state and survival in ischemia-reperfusion injury. To test this, we developed a human iPSC-derived heart-on-chip that includes resident macrophages and replicates architectural and immune complexity of the native myocardium.

Materials and

Methods: : Human cardiomyocytes, cardiac fibroblasts, macrophages, and endothelial cells were generated from a single iPSC line using previously validated protocols. These cells were combined in physiologic ratios in a collagen-based hydrogel. As described previously by our group, this hydrogel was embedded in the HEART, a custom microfluidic device fabricated through PDMS soft lithography. Endothelial cells were then seeded in the vascular channel. Tissues were maintained under media flow for 3 days under media flow to promote tissue organization and vasculature formation. A schematic of this construction is shown in Fig. 1a.
Myocardial infarction-like effects were induced as described previously. Ischemia was induced by ceasing media supply and culturing HEARTs in a hypoxic ( < 1% O2) incubator for 3 hours. Reperfusion injury was simulated by culture under media flow in normoxic conditions. After MI, cell death was measured in the presence or absence of macrophages with a live:dead stain. In reperfusion, macrophage precursors were flowed into the device to simulate immune cell recruitment.
Fluorescence lifetime imaging microscopy (FLIM) was used to, label-free and in real-time, visualize the metabolism of the tissues in the HEART. Autofluorescence of NAD(P)H was assessed in ischemia and reperfusion to determine metabolic status of cells in the tissue.
mtKeima-expressing CMs were used to visualize mitophagic flux in these cells with MI. Cardiomyocytes were differentiated from this cell line and integrated into the tissue in the same manner as above. Ratiometric imaging of low-pH and high-pH fluorescence of mtKeima was employed to interrogate mitophagy.

Results, Conclusions, and Discussions:: Cardiac tissues formed in the HEART device demonstrated robust integration of all four cell types, including stable retention of macrophages throughout culture. Immunostaining showed that macrophages remained within the hydrogel compartment and were distributed among cardiomyocyte clusters (Fig. 1b). High-resolution Airyscan microscopy confirmed the presence of cardiomyocyte gap junctions (Fig. 1c), indicating preservation of key electrophysiological features.
Another device was seeded with hydrogel containing solely macrophages. When media containing macrophage precursors were introduced into the vascular channel, a number of these macrophage precursors invaded the tissue channel, mimicking immune cell recruitment from blood into tissue (Fig. 1d). Taken together, these results establish the HEART as a functional model to study both resident and circulating macrophages in engineered cardiac tissue.
In parallel, macrophages were added to 2D cardiomyocyte cultures. After 9-12 days, these cultures were stained with JC-1. In cardiomyocytes which were cocultured with macrophages for 12 days and 9 days, there were significant changes in mitochondria polarization. Green fluorescence, a measure of total mitochondria, decreased after 9 days in coculture, with slight recovery after another 3 days. Increases in the proportion of highly polarized mitochondria occurred in these cultures as well (Fig. 2b). These changes were visually apparent (Fig. 2c).
Our macrophage-inclusive human heart-on-chip platform allows for the real-time study of immune–metabolic interactions in the infarcted myocardium. This offers a powerful new approach for probing immune contributions to cardiac disease and could inform the development of macrophage-targeted therapies in cardiovascular medicine.

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